1. Technical Field
The present invention relates to beads that are easily identified and have various functions, a method for reading the beads, and a bead-reading apparatus.
2. Background Art
It is conventionally known that beads are used for nucleic acid detection. In addition, JP Patent Publication (Kokai) No. 6-300763 A (1994) discloses the use of fluorescent microbeads for immunoassay. Since beads with sizes on the order of microns are used as sites for a specific reaction between biopolymers, they are dyed in an organic solvent and then read or identified (discriminated) by a fluorescence microscope or a flow cytometer. However, bead identification is not easy, and it has been difficult to easily and accurately identify a large number of beads, particularly when the number of different kinds thereof ranges from the tens to the ten-thousands. Further, when a light source such as laser is used for excitation to read fluorescence, light leakage occurs, resulting in some background noises. Thus, accurate reading cannot be performed.
Particles with sizes on the nano-order such as semiconductor nanoparticles and metal nanoparticles are gaining attention as labeling means that can be used alternatively to organic coloring or fluorescence agents. The nanoparticle of the present invention may be a particle having a particle size of 10 nm or less, which is generally called a “quantum dot” or a “nanodot.” The particle size thereof is preferably 1 to 5 nm. As kinds of materials used to form nanoparticles, known are metals such as gold, silver, palladium, and copper, semiconductors such as elemental semiconductors (Si, Ge, etc.) and compound semiconductors (GaAs, CdS, etc.), metal oxides such as titan oxide and tin oxide and chalcogenides.
Taking a semiconductor nanoparticle as an example, semiconductor nanoparticles of a grain size of 10 nm or less are located in the transition region between bulk semiconductor crystals and molecules. Their physicochemical properties are therefore different from those of both bulk semiconductor crystals and molecules. In this region, the energy band gap(=forbidden band) of a semiconductor nanoparticle increases as its grain size decreases, due to the appearance of quantum-size effects. In addition, the degeneracy of the energy band that is observed in bulk semiconductors is removed and the orbits are dispersed. As a result, the lower-end of the conduction band is shifted to the negative side and the upper-end of the valence band is shifted to the positive side.
Semiconductor nanoparticles can be easily prepared by dissolving equimolar amounts of precursors of Cd and X (X being S, Se or Te). This is also true for manufacturing CdSe, ZnS, ZnSe, HgS, HgSe, PbS, or PbSe, for example. However, the semiconductor nanoparticles obtained by the above method exhibit a wide grain-size distribution and therefore cannot provide the full advantages of the properties of semiconductor nanoparticles. Therefore, attempts have been made to attain a monodispersed distribution by using chemical techniques to precisely separate the semiconductor nanoparticles having a wide grain-size distribution immediately after preparation into individual grain sizes and extract only those semiconductor nanoparticles of a particular grain size. The attempts that have been reported so far include an electrophoresis separation method that utilizes variation in the surface charge of a nanoparticle depending on grain size, exclusion chromatography that takes advantage of differences in retention time due to differences in grain size, and a size-selective precipitation method utilizing differences in ability to disperse into an organic solvent due to differences in grain size. As a method that completely differs from the above methods, a size-selective optical etching method or the like has been reported, wherein the grain size of semiconductor nanoparticles is controlled by irradiating a solution of semiconductor nanoparticles with monochromatic light. Semiconductor nanoparticles obtained by these methods exhibit a spectrum with a relatively narrow wavelength-width peak.
As biologically specific reactions using beads with sizes on the micro-order as intermediates become more important from now on, there is a demand for developing a technology to easily identifying beads in order to make them usable. In particular, if large numbers of different kinds of beads, such as numbers ranging from the tens to the tens-thousands, are accurately and easily identified, the usability of beads is doubled. Additionally, when a light source such as laser is used for excitation and then fluorescence is read, light leakage occurs, so that accurate reading with the background light at zero level cannot be performed.